The goal of this proposal is identify the genes that correspond to the lupus susceptibility loci Sle1a and Sle1c and to determine the molecular and functional mechanisms by which their NZW alleles contribute to autoimmunity. We have evidence from congenic recombinants that 3 genes located in the Sle1a region contribute to lupus pathogenesis, for which we have identified two strong positional candidates: Pbx1, a ubiquitous homeobox transcription factor for Sle1a-1, and Sh2d1b / Sh2d1c, two duplicated genes that encode for adaptor proteins of the SLAM molecules, for Sle1a-2. In addition, Fcgr2b has already been associated to lupus in patients and in the NZM2410 model. In parallel, we have shown that an autoreactive T cell phenotype maps to the centromeric portion of Sle1c that we named Sle1c-2 (Sle1c-1 being Cr2). The analysis of congenic recombinants has identified two positional candidates for Sle1c-2, Erssg (estrogen-related receptor 3) and Ush2a (Usherin). To characterize these candidate genes, we have three specific aims. 1: To characterize the NZW allele of candidate genes for Sle1a-1, Sle1a-2, and Sle1c-2. We will use SNP genotyping to better define the critical interval for each locus and determine the SNP haplotype distribution within and around the candidate genes. We will characterize the NZW alleles for the four candidate genes in term of sequence and expression polymorphisms, and splice isoforms distribution. 2: To determine the mechanisms by which Sle1a-1, Sle1a-2 and Sle1c-2 and Fcgr2bNZW contribute to autoimmunity. We will use the in vivo and in vitro assays that we have developed to analyze the autoimmune phenotypes of the whole Sle1a and Sle1c intervals on the Sle1a and Sle1c congenic recombinants to pinpoint the cellular compartments and dissect the molecular mechanisms by which Sle1a-1, Sle1a-2 and Sle1c-2 induce autoimmune phenotypes. In addition, we will perform experiments to further characterize how the NZW allele of Fcgr2b contributes to autoimmunity.3: To validate the contribution of candidate genes to the Sle1a-1, Sle1a-2 and Sle1c-2 phenotypes. We will use lentiviral vectors (LV) to validate the candidate genes by either restoring a normal phenotype in the Sle1a or Sle1c sub-congenics, or by inducing a Sle1a or Sle1c phenotype in B6 mice, with bone-marrow cells LV-transduced with either the resistance or susceptibility alleles. The phenotypes and cellular compartments characterized in Aim 2 will be used as read-outs for the effects of over-expression of resistance or susceptibility alleles. This combined approach will identify the molecular and cellular mechanisms by which Sle1a and Sle1c-2 contributes to lupus pathogenesis, which we predict will be novel pathways regulating T cell tolerance to nuclear antigens.This project proposes to identify 3 genes that contribute to systemic lupus erythematosus, more specifically to the loss of tolerance to nuclear antigens in a spontaneous mouse model. Genetic analyses have provided a short list of candidate genes that we propose to characterize at the molecular and functional levels. These genes represent potential risk factors for human SLE patients and/or new disrupted pathways that are involved in lupus pathogenesis.